This continuation proposal is aimed at the design, preparation, and testing of advanced polymer beads for use as separation media in high- performance liquid chromatography (HPLC). These media will be designed to provide unmatched performance as a result of optimized physical properties and both bulk and surface chemistries. Our primary target is to develop very efficient polymeric stationary phases. Libraries of shape templates will be prepared, which use will enable the preparation of uniform 3 mu m beads with high porosity, well-defined regular surfaces, and tunable chemistry. Temperature gradients will be used during the staged templated suspension polymerization process to suppress the occurrence of micropores that appear to be the major problem of current polymer-based stationary phases. Synergistic effect of higher temperature and solvent that facilitates changes in conformation of polymer chains will be studied in detail and applied to the production of porous polymer beads with controlled topology of functionalities on the pore surface. Staying ahead of the current trend of rapid decrease in size of samples available for separations, our research will push even further the miniaturization of polymer-based separation technology using microcolumns and packed capillaries. Specific targets include functional beads and techniques for the separations of new generations of drugs as well as other biologically active compounds that will result from the discoveries of drug targets derived from the human genome. In particular, polymeric beads will be prepared with properties optimized for enantioselective chromatography. Advanced polymeric chiral stationary phases will be designed and optimized using the means of both computational and experimental combinatorial chemistry. First, methods of molecular docking will be used to explore virtual libraries of potential selectors and to facilitate the discovery of leads. Only more focused libraries will be then prepared in the laboratory. The chemistry of linkers will also be explored with the aim to reduce non-specific interactions and explore multiplicative as well as synergistic effects. The rational design of linkers, as well as newly prepared polymeric supports will enable the very efficient and/or selective separation of chiral drugs and their metabolites in addition to a number of other important enantiomers. This work will also contribute to the basic understanding of the effects of all of the critical elements of enantioselective separation media - the support, the linker, and the selector - on the recognition process.